Every individual cell is unique and can differ by size, state, protein expression and can also exhibit distinct genomic profiles, even within the same tissue. These dissimilarities are important, especially in understanding rare diseases/cell events which are often masked out in population average measurements. For instance, detecting circulating tumour cells (CTC) in blood circulation is a potential non-invasive method for cancer prognosis. Beyond enumeration, which is correlated to tumour load, CTC characterization is expected to guide therapeutic selection for personalized care of patients. Single cell analyses can provide the critical piece of information that helps identify these changes. Conventional methods of extraction by pipetting and processing cells are tedious and error prone, especially when target cells are low in quantity and/or purity. Automated fluorescence cell sorters require millions of cells and introduce significant cell losses, which makes them unsuitable for this application.
Single cell analysis is gaining traction due to the potential benefits of addressing cellular heterogeneity. Important variations present in a small number of cells can be masked when analysis is performed on a cell population. Recovery and characterization of individual cells is therefore an essential part of many current studies in oncology and regenerative medicine. As medical practices gear towards personalized and targeted therapy; understanding the diversity and genetic makeup of cell populations will aid practitioners to better make clinical judgments. This will also speed up the drug design and screening processes, if we are able to identify the key mutations associated with the diseases. The ability to accurately select, manipulate and process single cells in a high throughput fashion is useful to obtain a sufficient sample size for understanding cellular heterogeneity.
Conventional bench top methods of single cell sorting and preparation include serial dilution and manual pipetting of cells under a microscope. To achieve single cell resolution using serial dilution results in large errors which are random and difficult to spot. Manual pipetting of cells is a fairly low throughput technique and operations are tedious, meaning that it will not be suitable for processing the hundreds of cells required to generate sufficient data points to characterize cellular subpopulation diversity patterns. Current semi-automated methods of isolation involving micropipette selection and using micro-manipulators or laser capture dissection are also low throughput processes, with the user having to navigate to the cells of interest individually to perform the isolation process.
Fluorescence activated cell sorter (FACS) has the ability to achieve single cell separation through optical interrogation. However, these systems require a sizeable amount of starting material and are less suited for rare cell events due to large cell losses. The FACS system is also a fairly expensive tool and is usually shared among several users, thereby risking contamination from sample to sample which might result in false positive outcomes in a subsequent genomic analysis. This limits the usefulness of the FACS system for rare specimens.
Other forms of cell sorters have been proposed, most notably using microsystems. These types of systems are favored due to low sample input requirements and allow a fairly reasonable throughput. These devices are also cheaper and allow for a single use to prevent cross contamination. Devices that make use of magnetic separation in a continuous flow had been shown to be highly efficient with enrichment as high as 5000 fold to discriminate against different cellular sub populations. The main limitation of such systems is that they do not allow for the recovery of single cells, which limits certain downstream analyses such as next generation sequencing (NGS), which is a useful tool to probe genetic variations on a large scale. Other sorting techniques which are considered gentle to the cells include separation through deterministic lateral displacement or by altering the laminar flow characteristics in micro flows. However, the velocities at which samples are traversing across the microchannels are too fast to make an accurate measurement of the fluorescence intensities without sophisticated equipment, which also makes the recovery of single cells difficult.
For systems that address sorting/recovery at the single cell level, these include micro patterning of substrates for single cell deposition, physical trapping using microfluidics, inkjet printing of cells and dieletrophoretic (DEP) forces that manipulate cells at its active probing region. For instance, it will be difficult to use micropatterning for specific cell recovery to perform DNA extraction for molecular analysis without affecting adjacent adhered cells. Sophisticated controls for systems using dielectrophoresis (DEP) forces and inkjet printing of cells are additional costs which are significant. For example, a major limitation of the Fluidigm™ single cell sample preparation system, which uses passive physical trapping, requires target cells of higher than 1.1% frequency because there are only 96 wells within the system. If the frequency of the desired cells is lower than 1.1%, then there is a good probability that none of the target cells will be extracted out of the run. Even so, much of the sample within the system is wasted as most are not the target cells, unless the input is relatively pure. Therefore, there remains a need for a system that can enable the speedy recovery of cells, but which is also sufficiently flexible to enable archival of the cells or enables the use of said cells in various downstream applications.